PROJECT SUMMARY The immune-mediated, chronic inflammation in inflammatory bowel disease (IBD) leads to flares that often require patients to be treated with life-long biologic therapy. Recently, efforts in IBD disease management have focused on the potential of regulatory T (Treg) cell-based therapy as an alternative to traditional treatments. Treg cells limit inflammatory responses to both foreign and self-antigens and can become further differentiated into potently suppressive effector Tregs (eTregs) upon exposure to immune stimuli. eTregs, especially those that produce the immunosuppressive cytokine interleukin 10 (IL-10), play a critical, mitigating role in both human and murine colitis. However, the signals that induce and functionally enhance this subset are not well understood, largely because they are limited in number and there have been no suitable protocols to differentiate them in vitro. Here, we have identified a signal that enhances the expansion and maintenance of IL-10+ eTregs in vivo and in vitro. Death receptor 3 (DR3 or TNFRSF25) is a tumor necrosis factor receptor superfamily (TNFRSF) member that is constitutively expressed by activated T cells and has been found to both enhance and suppress the immune response. We have found that treatment with an agonist antibody to DR3 (aDR3) significantly enhances the proliferation and cell survival specifically of IL-10+ eTregs. Additionally, treatment greatly expands total Treg cells in many tissues, but the effect is most profound in the intestine. To our knowledge, the observation that aDR3 expands IL-10 producing eTregs has not yet been described. This proposal will determine the mechanisms by which DR3 signaling induces increased intestinal Tregs (Aim 1), as well as the potential of aDR3 as a therapeutic in the treatment of colitis (Aim 2). We will utilize a novel transgenic mouse model that lacks DR3 specifically in Tregs to understand the role that DR3 signaling plays in intestinal Treg function in both the steady state and in the inflamed colon. By using our IL-10 and Foxp3 dual reporter mouse, we will be able to stratify Treg populations by IL-10 competency, which will allow us to characterize the mechanisms by which DR3 differentially enhances Treg cell subsets. Our central hypothesis is that the effect of DR3 signaling is two-fold in that it: (1) primes Treg cells for an effector phenotype; and (2) enacts a specific program that impacts eTreg survival, stability, and function. Understanding how this pathway augments Treg cell function will have broad- reaching implications not only in the field of IBD, but also in the management of a number of immune-mediated diseases.